Telomeres are genetic elements located at the ends of all eukaryotic chromosomes which preserve genome stability and cell viability by preventing aberrant recombination and degradation of DNA. In humans, the telomeric sequence is composed of 10-20 kilobases of TTAGGG repeats (Blackburn, (1991) Nature vol. 350 pp 569-573). There is increasing evidence that gradual loss of telomeric repeat sequences may be a timing (“clock”) mechanism limiting the number of cellular divisions in normal somatic cells (Harley et al., (1990) Nature, vol. 345, pp. 458-460). In contrast, immortal cells are capable of maintaining a stable telomere length by upregulating or reactivating telomerase, a ribonucleoprotein enzyme that is able to add TTAGGG repeats to the ends of chromosomes (Greider and Blackburn, (1989) Nature, vol. 337, pp. 331-337; Morin, (1989) Cell, vol. 59, pp. 521-529).
Telomerase activity has been detected in 85% of primary human tumors tested from a variety of tissue types (Kim et al., (1994) Science, vol. 266, pp. 2011-2015; Shay and Bacchetti, (1997) European Journal of Cancer, vol. 33, No. 5, pp. 787-791). The detection of high telomerase activity in human cells or tissues almost always correlates with indefinite proliferation capability (immortalization). U.S. Pat. No. 5,648,215 describes the presence of telomerase activity in somatic cells as indicative of the presence of immortal cells, such as certain types of cancer cells, which can be used to make that determination even when the cells would be classified as non-cancerous by pathology. In addition, when telomerase assays are performed on certain normal stem cell compartments (e.g. bone marrow) or in regions of highly proliferative cells (e.g. hair follicles and crypt gut epithelial cells), and a high level of telomerase activity is detected, this can be indicative of active regenerating tissue.
Methods for detecting telomerase activity, as well as for identifying compounds that regulate or affect telomerase activity, have been described. See PCT patent publication No. 93/23572, U.S. Pat. Nos. 5,629,154, 5,648,215, 5,645,986, 5,695,932 and 5,489,508. Each of the foregoing patent publications is incorporated herein by reference.
For example, U.S. Pat. Nos. 5,629,154; 5,863,726 and 5,648,215 describe in detail the preparation of a cell extract using a detergent lysis method and the analysis of telomerase activity by the Telomeric Repeat Amplification Protocol (TRAP assay). The telomerase activity assays described therein involve the extension of a synthetic nucleic acid substrate derived from the telomere sequence which serves as a primer. This primer is added together with unlabelled dideoxynucleotides to a sample, e.g. a cell extract suspected of containing telomerase whereby the primer is specifically elongated by the telomerase.
The extended primer may be detected directly by a number of methods. The primer may be directly attached to an anchor group which can be immobilized on a solid phase using any known method. Examples of suitable anchor groups are biotin which can bind with high affinity binding to an immobilized partner such as avidin or streptavidin. Detection of the immobilized primer can be measured by either incorporation of a radioactive deoxynucleotide or by the binding of a sequence specific labeled probe to the extended primer.
The extended substrate may also be detected after it is replicated in a primer extension reaction, such as the polymerase chain reaction (PCR) a radioactive deoxyribonucleoside triphosphate (dNTP) for labeling any telomerase-extended substrate. The reaction mixture is subsequently separated by gel electrophoresis and the pattern of bands visualized. Because telomerase stalls and can release the DNA after adding the first G in the 5′-TTAGGG-3′ telomeric repeat, the characteristic pattern of products on the gel is a six nucleotide ladder of extended oligonucleotide substrates. The phase of the repeats depends on the 3′-end sequence of the substrate; telomerase recognizes where the end is in the repeat and synthesizes accordingly to yield contiguous repeat sequences.
However, these detection methods of the state of the art have some disadvantages. The level of telomerase enzyme in human cells containing the enzyme is very low in the order of 10-100 molecules per cell. Therefore, the sensitivity of a direct detection method without an amplification step may be too low for routine applications since quantities of extracts containing >107 cells typically have to be used for direct detection. Therefore this method cannot be used to examine primary tumor material which is only available in a small amount.
In addition, the gel images of the telomerase products are poor; often appearing as a smear rather than distinct bands, and quantitation of these bands is difficult, imprecise, and inaccurate.
A number of factors contribute to these issues, but the most important factor is likely the impurity of the telomerase extract itself Telomerase is present at roughly 10 ppb (w/w protein) i.e. 0.000001%, in a crude cell extract from telomerase positive cells. In a tissue which contains a mixture of telomerase positive and negative cells, the relative abundance is even lower. The 100 million-fold excess of other proteins that are released upon disruption of the cells into the reaction mixture can interfere with the telomerase activity assay by non binding to the primers, by interference with the activity of the telomerase or the taq (PCR) polymerase enzyme, by degradation of the telomerase or taq enzyme, primers, or probes, or by consumption or binding to other components in the reaction mixture (e.g. dNTPs and metal ions).
Accordingly, neither method is suitable for cells with very low levels of telomerase enzyme or for very small tissue samples. Moreover, neither method allows a high sample throughput. The methods are not suitable for automation as is necessary for example for routine analysis of tissue samples for analysis of telomerase modulators. Identification of a method of isolating the telomerase enzyme and routine analysis for telomerase activity would be beneficial.